Breast cancer is one of the leading causes of premature death in women and development of chemoprevention and chemotherapeutic interventions wilt require understanding of the important steps that lead to tumor formation, growth and metastasis. Estrogen receptor (ER)-positive and -negative breast cancer cells have been extensively used as models for studying signaling pathways that are important for breast tumor growth. Both 17beta-estradiol (E2) and polypeptide growth factors (GFs) have been identified as important mitogens for breast cancer cell growth. We hypothesize that ERalpha-dependent activation of nongenomic phosphatidyl inositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways by E2 are important for breast cancer cell proliferation, and the proposed studies will investigate the mechanisms of non-genomic activation by E2. Aim 1 will focus on the role of ERalpha in activation of PI3-K and MAPK using ER-positive breast cancer cells (MCF-7 and ZR-75) and ER-negative (CHO and COS) cells as models. The domains of ERalpha required for activation of kinases and the importance of cell context will be determined in transient transfection studies. The mechanisms of non-genomic action of E2 and the role of specific kinases in mediating activation of PI3-K and MAPK will be investigated in Aim 2. The studies will also determine the biological significance of direct ERalpha interactions with Src-SH2, G proteins, the p85 regulatory subunit of PI3-K and the IGF-1 receptor. Aim 3 will focus on downstream mechanisms of ERalpha-dependent activation of c-fos by kinases. Dominant negative p85, MAPKK and Src expression plasmids transiently or stably transfected into breast cancer cells will be used to investigate the contribution of the MAPK and PI3-K pathways to the mitogenic activity of E2 in breast cancer cells (in vitro) and mammary tumors in athymic nude mice bearing breast cancer cell xenografts. The in vitro mechanistic studies will also be complemented by research on the contributions of kinase activation by E2 on cell proliferation and tumor growth and thereby define potential therapeutic targets for treating breast cancer.